The present invention relates to genetic engineering, particularly to compositions and methods for altering the normal pattern of expression associated with a particular promoter-driven construct in plants using nuclear matrix attachment regions.
Extensive literature exists on the potential role of matrix attachment region (MAR) DNA sequences in the regulation of eukaryotic gene expression (see, for example, Mirkovitch et al. (1984) Cell 39:223-232; Stief et al. (1989) Nature 341:343-345; Bode et al. (1992) Science 255:195-197; Spiker and Thompson (1996) Plant Physiol. 110:15-21). MAR sequences (also called scaffold attachment region, or SAR, sequences) are examples of elements that are thought to play a role in the regulation of transcription. Early work established that MAR sequences must be incorporated into the host genome to have their effect (Stief et al. (1989) Nature 341:343-345). These regions of highly AT-rich DNA (more than 70%) have been shown to increase transgene expression in stably transformed animal cell lines (see, for example Stief et al. (1989) Nature 341:343-345; Phi-Van et al. (1990) Mol. Cell. Biol. 10:2302-2307; Klehr and Bode (1991) Biochemistry 30:1264-1270; Poljak et al. (1994) Nucleic Acids Res. 22:4386-4394; Kalos and Fournier (1995) Mol. Cell. Biol. 15:198-207) and transformed plants (see, for example, van der Geest et al. (1994) Plant J. 6:413-423; Schxc3x6ffl et al. (1993) Transgenic Res. 2:93-100; Allen et al. (1993) Plant Cell 5:603-613; Mlynxc3xa1rovxc3xa1 et al. (1994) Plant Cell 6:417-426 and (1995) Plant Cell 7:599-609; and Spiker et al. (1995) J. Cell Biochem. 21B: 167). Decreased transformant-to-transformant variability in expression with the use of MAR sequences has been reported less frequently (see Stief et al. (1989) Nature 341:343-345; Breyne et al. (1992) Plant Cell 4:463-471; van der Geest et al. (1994) Plant J. 6:413-423; Mlynxc3xa1rovxc3xa1et al. (1994) Plant Cell 6:417-426). This position-independent expression has been attributed to insulation of foreign DNA inserts from position effects, possibly by protecting the DNA insert from interfering effects of adjacent chromatin enhancers or silencers, or by inhibiting methylation. Additionally, copy-number dependence (i.e., increased levels of expression with increased copies of the transgene) with the use of MAR sequences has been infrequently reported for transformed animal cell lines (see Stief et al. (1989) Nature 341:343-345) and transformed plants (vander Geest et al. (1994) Plant J. 6:413-423).
MAR sequences serve to attach chromatin loop domains to the nuclear matrix fiber, forming the boundaries for these DNA loops (Gasser et al. (1989) Int. Rev. Cytol. 119:57-96; Laemmil et al. (1992) Curr. Opin. Genet. Dev. 2:275-285; Dorer and Henikoff(1994) Cell 77:993-1002). Their exact role in eukaryotic gene expression is not known, though several hypotheses have been proposed. Early models suggested that incorporation of foreign DNA into the host genome occurs randomly in the absence of MAR sequences. Hence, if incorporation occurs within a transcriptionally inactive chromatin domain, the foreign DNA takes on an inactive chromatin structure, thus reducing the potential for transcription of the foreign DNA. If incorporation occurs within a transcriptionally active chromatin domain, the transgene takes on the active chromatin structure, thus increasing the potential for transcription of that DNA. If the foreign DNA is flanked by MAR sequences, however, incorporation into an active or inactive region results in the formation of an independent domain, which itself may assume an active or inactive chromatin state.
The functional importance of the independent domain is that the foreign DNA insert is isolated from the effects of the chromatin around it, hence contributing to the suppression of gene silencing and position effects, and overall enhancement of expression. This model is oversimplified, however, as it cannot explain persistent variation in expression of low-copy transformants and inconsistencies in copy number-dependent transgene expression (see Spiker and Thompson (1996) Plant Physiol. 110: 15-21).
Others have proposed that MAR sequences form nucleation points for DNA unwinding (Bode et al. (1992) Science 225:195-197); that MAR sequences form sites of nucleation for HMG proteins to displace H1 histones, allowing highly coiled chromatin fibers to unwind (Kas et al. (1993) EMBO J. 12:115-126); that MAR sequences stabilize chromosomal topology arising as a consequence of hyperacetylation of histone cores (Schlake et al. (1994) Biochemistry 33:4197-4206); and that MAR sequences stimulate transgene expression by reducing the severity of homology-dependent gene silencing (Spiker and Thompson (1996) Plant Physiol. 110:15-21).
To date the predominant investigatory focus has been on the use of MAR sequences to enhance transgene expression. Little is known about other potential roles for those sequences such as their ability to alter normal patterns of expression. Such changes might include a modification of expression so that, when a transgene is operably linked to a promoter with a characteristic pattern of expression (i.e. constitutive) the addition of MAR elements alters this pattern of expression, generating a promoter that drives expression in a tissue-preferred or tissue localized manner.
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. When continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. In contrast, when gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. When expression in specific tissues or organs are desired, tissue-preferred promoters may be used.
While a number of promoters are readily available and are frequently used in research involving recombinant DNA technology, these promoters are primarily limited to their native functional character or pattern of expression, i.e., constitutive, inducible, etc. Methods by which these promoters, or the pattern of expression exhibited by these promoters can be manipulated to generate an altogether different pattern of expression have thus far been unreliable. There is great value in the ability to manipulate the expression pattern of any promoter by simply genetically engineering into it the capacity to express a coding sequence behind its control in a wholly different manner. Thus, this invention is drawn to the use of nuclear matrix attachment regions (MAR) DNA sequences to alter the normal pattern of expression associated with a particular promoter-driven construct and thereby generating a promoter capable of driving expression of a heterologous nucleotide sequence in a manner which satisfies the needs of an individual investigator.
A DNA construct comprising matrix attachment region (MAR) sequences having altered expression patterns is provided. The invention further encompasses a method of altering the characteristic expression pattern associated with a promoter-driven construct by using MAR sequences.
One aspect of the present invention is a DNA construct comprising an expression cassette having, in the 5xe2x80x2-to-3xe2x80x2 direction, a nucleotide sequence or gene of interest operably linked to the transcription initiation region or promoter, a transcription and translation termination region, and a matrix attachment region DNA sequence positioned either 5xe2x80x2 to the transcription initiation region, 3xe2x80x2 to the termination region, or in both 3xe2x80x2 and 5xe2x80x2 positions. Preferably, the expression cassette is flanked by the MAR DNA sequences positioned both 5xe2x80x2 to the transcription initiation region and 3xe2x80x2 to the termination region. This DNA construct may be assembled within the backbone of any conventional vector.
A second aspect of the present invention is a method for modifying or altering the characteristic expression patterns associated with a particular promoter-driven construct in plants by flanking the construct with at least one matrix attachment region (MAR) DNA sequence. This method comprises transforming a regenerative plant cell with the DNA construct of this invention using conventional transformation methods known in the art. More preferably the method comprises altering the native constitutive expression pattern of a promoter-driven construct to exhibit or be capable of expression in a tissue localized manner. Even more preferably the promoter-driven construct whose expression is altered comprises the small synthetic promoter, Rsyn7.
The present invention also provides for stably transformed plants, which comprise the DNA construct according to the invention, that exhibit tissue localized expression of a heterologous nucleotide sequence as a result of the attachment of the MAR DNA sequences to the 5xe2x80x2 and 3xe2x80x2 ends of the construct. Seeds of such plants are also provided.